Gamma-linolenic acid (GLA; γ-linolenic acid; cis-6, 9, 12-octadecatrienoic acid) is an important intermediate in the biosynthesis of biologically active prostaglandin from linoleic acid (LA). Additionally, GLA is recognized as an essential ω-6 polyunsaturated fatty acid (PUFA) having tremendous clinical and pharmaceutical value. For example, GLA has been shown to reduce increases in blood pressure associated with stress and to improve performance on arithmetic tests. GLA and dihomo-γ-linolenic acid (DGLA, another ω-6 PUFA) have been shown to inhibit platelet aggregation, cause vasodilation, lower cholesterol levels and inhibit proliferation of vessel wall smooth muscle and fibrous tissue (Brenner et al., Adv. Exp. Med. Biol. 83:85-101 (1976)). Administration of GLA or DGLA, alone or in combination with eicosapentaenoic acid (EPA, an ω-3 PUFA), has been shown to reduce or prevent gastrointestinal bleeding and other side effects caused by non-steroidal anti-inflammatory drugs (U.S. Pat. No. 4,666,701). Further, GLA and DGLA have been shown to prevent or treat endometriosis and premenstrual syndrome (U.S. Pat. No. 4,758,592) and to treat myalgic encephalomyelitis and chronic fatigue after viral infections (U.S. Pat. No. 5,116,871). Additionally, GLA is also used in products relating to functional foods (nutriceuticals), infant nutrition, bulk nutrition, cosmetics and animal health.
Although seeds of many plants contain GLA, the most common commercial sources of GLA are evening primrose (Oenothera biennis L.; containing 5-9% GLA of total seed oil), black currant (Ribes nigrum; containing 14% GLA of total seed oil) and borage (Borago officinalis L.; containing 17-25% GLA from a total seed oil content of 28-38%) (Simon, J. E. et al., In Advances In New Crops; Janick, J. and J. E. Simon, Eds.; Timber: Portland, Oreg. (1990); p 528). There are several disadvantages associated with production of GLA from these sources, however. First, plant oils tend to have highly heterogeneous oil compositions that can require extensive purification to separate or enrich one or more of the desired PUFAs. Natural sources are also subject to uncontrollable fluctuations in availability (e.g., due to weather or disease); and, crops that produce PUFAs often are not competitive economically with hybrid crops developed for food production. Specific challenges associated with large-scale commercial production of borage are encountered because of the plants' indeterminate vegetative growth, lack of concentrated flowering and seed set, non-uniform seed maturation, susceptibility to a wide range of insects and disease pests, and poor ability to compete with weeds.
To overcome these problems, microorganisms have been investigated as an alternative source of GLA and other PUFAs. Specifically, many microorganisms (including algae, bacteria, molds and yeast) can synthesize oils in the ordinary course of cellular metabolism. Thus, oil production involves cultivating the microorganism in a suitable culture medium to allow for oil synthesis, followed by separation of the microorganism from the fermentation medium and treatment for recovery of the intracellular oil. Microbial production of GLA has been investigated, although these efforts have been minimal in comparison to the work focused on the microbial production of longer-chain PUFAs such as arachidonic acid (ARA), EPA and docosahexaenoic acid (DHA). Some suitable strains have been proposed as perspective GLA producers, including Mortierella ramanniana, Mucor sp., Cunninghamella japonica and Entomophthora exitalis. Currently, however, only Japan is producing GLA commercially using the fungus Mortierella (Ratledge, C. Trends Biotech. 11:278-284 (1993)). Thus, there remains a need for an appropriate microbial system for economical production of commercial quantities of GLA.
One class or microorganisms that has not been previously examined as a production platform for PUFAs such as GLA are the oleaginous yeast. These organisms can accumulate oil up to 80% of their dry cell weight. The technology for growing oleaginous yeast with high oil content is well developed (for example, see EP 0 005 277B1; Ratledge, C., Prog. Ind. Microbiol. 16:119-206 (1982)) and may offer a cost advantage compared to commercial micro-algae fermentation for production of PUFAs. Whole yeast cells may also represent a convenient way of encapsulating PUFA-enriched oils for use in functional foods and animal feed supplements.
Despite the advantages noted above, oleaginous yeast are naturally deficient in PUFAs, since naturally produced PUFAs in these organisms are limited to 18:2 acids (and, less commonly, 18:3 acids). Thus, the problem to be solved is to develop an oleaginous yeast that accumulates oils enriched in GLA. Toward this end, it is necessary to introduce desaturases that allow for the synthesis and accumulation of GLA in oleaginous yeast. Although advances in the art of genetic engineering have been made, such techniques have not been developed or optimized for oleaginous yeast; and, one must overcome problems associated with the use of these particular host organisms for the production of GLA.
Applicants have solved the stated problem by engineering strains of Yarrowia lipolytica that are capable of producing over 25% and 34% GLA in the total lipids, respectively. Additional metabolic engineering and fermentation methods are provided to further enhance GLA productivity in oleaginous yeast.